Ligand specificity and evolution of liver X receptors.

Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, United States.

Abstract

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.

Concentration-response curves for activation of LXRs by T-0901317 and oxysterols. The ordinate represents activation of LXR, relative to vehicle control, and normalized to the maximal activator (5 μM T-0901317 for mLXRα, mLXRβ, and zfLXR; 10 μM T-0901317 for hLXRα, hLXRβ, xlLXR, and xtLXR; 50 μM androstenol for ciLXR). The compounds tested were T-0901317 (●), 24(S)-hydroxycholesterol (○), 25-hydroxycholesterol (□), 24(S),25-epoxycholesterol (⋄), and 24-ketocholesterol (Δ). (A-D,F,G) hLXRα, hLXRβ, mLXRα, mLXRβ, xtLXR, and zfLXR show very similar patterns of activation by the five compounds. (E) For xlLXR, the efficacy of the oxysterols was much lower than that of T-0901317. (H) ciLXR was not activated by T-0901317 but was activated by three of the oxysterols.

Analysis of Xenopus tropicalis bile. (A) ESI/MS analysis of Xenopus tropicalis bile. From an extensive library generated from analysis of many vertebrate bile specimens, the major ions are annotated with probable matches indicating the number of carbon atoms (24 or 27), whether the compound is a bile acid or bile alcohol, number of hydroxyl groups, as well as absence or presence of conjugation (taurine for acids, sulfate for alcohols). The dominant ion corresponds to the m/z ratio expected for a penta-hydroxylated C27 bile alcohol sulfate (e.g., cyprinol sulfate); in addition, m/z ratios consistent with trihydroxylated unconjugated and taurine-conjugated C24 and C27 bile acids are also present. (B) HPLC analysis of X. tropicalis bile shows the likely presence of taurocholic acid (13.9 min), taurochenodeoxycholic acid (24.0 min), and taurodeoxycholic acid (28.3 min).

Molecular modeling studies of human LXRβ and Ciona LXR. HIPHOP alignments of 6-formylindolo-[3,2-b]-carbazole with (A) 24(S),25-epoxycholesterol and (B) T-0901317. Green indicates hydrogen bond acceptor while cyan denotes hydrophobic features. (C) HIPHOP model for ciLXR based on multiple active and inactive molecules. (D) HIPHOP model for hLXRα based on multiple active and inactive molecules. The color schemes for both (C) and (D) are as follows: epoxycholesterol – grey; 6-formylindolo-[3,2,b]-carbazole – yellow; hydrogen bond acceptor – green; hydrophobic features – cyan; hydrogen bond donor – purple; excluded volumes – grey. (E) Docking of epoxycholesterol into the predicted binding pocket of ciLXR generated by a homology model. The orientation of epoxycholesterol is flipped 180° relative to that seen in a crystal structure of hLXRβ []. Epoxycholesterol has good complementarity to the pocket, but the hydrogen bond interaction of the 3-hydroxyl group of epoxycholesterol seen in the ηυμα ΛΞPβ crystal structure is not present. F, Docking of 25-hydroxycholesterol into the predicted binding pocket of ciLXR generated by a homology model. The 3-hydroxyl group of 25-hydroxycholesterol forms a hydrogen bond with Gln-314 and the tail part of the 25-hydroxycholesterol has extensive hydrophobic contacts with the binding pocket.